1. Field of the Invention
The present invention relates to polymerase chain reactions generally and, more particularly, but not by way of limitation, to a novel continuous polymerase chain reaction process having multiple temperature stations.
2. Background Art
In the field of genomics, and other disciplines using molecular biology, the polymerase chain reaction (PCR) protocol is essential. It is an amplification technique that utilizes three basic temperatures to amplify DNA. In such a protocol, the DNA is first brought to 96xc2x0 Centigrade to denature the DNA, causing it to xe2x80x9cunwindxe2x80x9d from the standard double helix to single strands. The denaturing process requires exposure to 96xc2x0 Centigrade for approximately 15 seconds.
Next in the protocol, the DNA is exposed to a temperature of 50-55xc2x0 Centigrade to anneal the single strands, normally in the presence of defined primers. Again, approximately only 15 seconds at 50-55xc2x0 Centigrade is required for annealing. The next temperature is 72xc2x0 Centigrade. At this extension temperature, the two single strands form two double stranded helixes, thus resulting in a two-fold amplification. The extension temperature of 72xc2x0 Centigrade is only required for 30 seconds.
The foregoing temperature cycling doubles the amount of DNA on each cycle. After 25 to 35 cycles, non-measurable quantities of DNA now become readily detectable because of the power of PCR and its exponential amplification.
The current state-of-the-art techniques for thermocycling comprise two basic methods. One is a batch method, whereby a group of PCR reaction plates is physically moved from one water temperature bath to another. The second, and more popular, method is the use of thermocycling instrumentation using Peltier thermoelectric devices to change the temperature of an individual PCR plate.
The Peltier thermoelectric device is clean and efficient; however, it process only one plate at a time. While the latter feature is an advantage for small operators, it is a disadvantage in high volume operations. High volume laboratories will have bench tops with many thermocyclers side by side. At a cost of $5,000-6,000 each, a considerable investment is required, particularly since the nature of genomic testing requires a high volume of testing.
Another disadvantage of the thermocycling instrument is the time required to move from one temperature to the next. At present, the popular Peltier devices can only change temperature at a rate of about 3 Centigrade degrees per second. The change from 96xc2x0 Centigrade to 50xc2x0 Centigrade requires 15 seconds transient time plus the 15 seconds at the annealing temperature. From 50xc2x0 Centigrade to 72xc2x0 Centigrade requires 7 seconds transient time plus the 30 second extension time. From 72xc2x0 Centigrade to 96xc2x0 Centigrade requires 8 seconds. Thus, for the 60 seconds of protocol time, an additional 30 seconds is required for transient time. This adds 50 percent to the overall time cycle. While insignificant on a single cycle, the time is an additional 12 minutes per plate on a 25 cycle protocol and 17 minutes per plate on a 35 cycle protocol.
The batch method of inserting a stack of plates into separate water baths decreases the temperature transient time. While the batch method is suitable for batches of large numbers of plates, the set up and handling time makes running small batches less attractive.
Accordingly, it is a principal object of the present invention to provide a PCR process that greatly reduces temperature transient times.
It is a further object of the invention to provide such a process that is economical for either a small or a large number of DNA samples.
It is another object of the invention to provide such a process that is easily implemented.
A further object of the invention is to provide such a process that is continuous.
Other objects of the present invention, as well as particular features, elements, and advantages thereof, will be elucidated in, or be apparent from, the following description and the accompanying drawing figures.
The present invention achieves the above objects, among others, by providing, in a preferred embodiment, a method of performing a reagent protocol using polymerase chain reaction, comprising: indexing patterns of reagent wells on a continuous basis through at least one step of reagent addition to said reagent wells; and then indexing said patterns of reagent wells on a continuous basis through a plurality of individual heat transfer stations, whereby at each of said individual heat transfer stations, said patterns of reagent wells are subjected to a unique temperature change to cause one amplification step, with said plurality of individual heat transfer stations providing total amplification required for said protocol.